title: Deficiency of ALADIN impairs redox homeostasis in human adrenal cells and inhibits steroidogenesis.
abstract: Triple A syndrome is a rare, autosomal recessive cause of adrenal failure. Additional features include alacrima, achalasia of the esophageal cardia, and progressive neurodegenerative disease. The AAAS gene product is the nuclear pore complex protein alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN), of unknown function. Triple A syndrome patient dermal fibroblasts appear to be more sensitive to oxidative stress than wild-type fibroblasts. To provide an adrenal and neuronal-specific disease model, we established AAAS-gene knockdown in H295R human adrenocortical tumor cells and SH-SY5Y human neuroblastoma cells by lentiviral short hairpin RNA transduction. AAAS-knockdown significantly reduced cell viability in H295R cells. This effect was exacerbated by hydrogen peroxide treatment and improved by application of the antioxidant N-acetylcysteine. An imbalance in redox homeostasis after AAAS knockdown was further suggested in the H295R cells by a decrease in the ratio of reduced to oxidized glutathione. AAAS-knockdown SH-SY5Y cells were also hypersensitive to oxidative stress and responded to antioxidant treatment. A further impact on function was observed in the AAAS-knockdown H295R cells with reduced expression of key components of the steroidogenic pathway, including steroidogenic acute regulatory and P450c11β protein expression. Importantly a significant reduction in cortisol production was demonstrated with AAAS knockdown, which was partially reversed with N-acetylcysteine treatment.
  
  
  CONCLUSIONS
  Our in vitro data in AAAS-knockdown adrenal and neuronal cells not only corroborates previous studies implicating oxidative stress in this disorder but also provides further insights into the pathogenic mechanisms in triple A syndrome.
authors.affiliation: Centre for Endocrinology, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Prasad
authors.forename: L A
authors.initials: LA
authors.lastname: Metherell
authors.forename: A J
authors.initials: AJ
authors.lastname: Clark
authors.forename: H L
authors.initials: HL
authors.lastname: Storr
mesh_terms: D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000305:Adrenal Cortex Hormones / Q000378:metabolism*; D000309:Adrenal Insufficiency / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004931:Esophageal Achalasia / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism; D055785:Gene Knockdown Techniques; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D034741:RNA, Small Interfering; D013252:Steroid 11-beta-Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: C416506:AAAS protein, human; D000305:Adrenal Cortex Hormones; D000975:Antioxidants; D009419:Nerve Tissue Proteins; D028861:Nuclear Pore Complex Proteins; D016877:Oxidants; D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D000098682:Steroidogenic Acute Regulatory Protein; D011691:Puromycin; D013252:Steroid 11-beta-Hydroxylase; D005978:Glutathione